Effects of Capsular Polysaccharide amount on Pneumococcal-Host interactions

Among the many oral streptococci, Streptococcus pneumoniae (Spn) stands out for the capacity of encapsulated strains to cause invasive infection. Spread beyond upper airways, however, is a biological dead end for the organism, raising the question of the benefits of expending energy to coat its surface in a thick layer of capsular polysaccharide (CPS). In this study, we compare mutants of two serotypes expressing different amounts of CPS and test these in murine models of colonization, invasion infection and transmission. Our analysis of the effect of CPS amount shows that Spn expresses a capsule of sufficient thickness to shield its surface from the deposition of complement and binding of antibody to underlying epitopes. While effective shielding is permissive for invasive infection, its primary contribution to the organism appears to be in the dynamics of colonization. A thicker capsule increases bacterial retention in the nasopharynx, the first event in colonization, and also impedes IL-17-dependent clearance during late colonization. Enhanced colonization is associated with increased opportunity for host-to-host transmission. Additionally, we document substantial differences in CPS amount among clinical isolates of three common serotypes. Together, our findings show that CPS amount is highly variable among Spn and could be an independent determinant affecting host interactions.

Introduction Streptococcus pneumoniae (Spn, the pneumococcus) is the leading bacterial pathogen associated with deaths in children less than five years of age worldwide [1]. Virulent Spn are covered by a thick layer of capsular polysaccharide (CPS) [2]. In the absence of this layer, the organism is unable to cause the main invasive infections (pneumonia, sepsis, meningitis) responsible for its burden on public health [3]. The species expresses at least 100 'types' of structurally and immunologically distinct CPSs. While CPS is the basis of all currently licensed Spn vaccines, only limited CPS types are included as antigens. Thus, the heterogeneity of Spn capsule limits the effectiveness of current prevention strategies.
Because of its importance as a determinant of virulence and vaccine antigen, there has been much focus on understanding the contribution of CPS to the biology of the pneumococcus [4]. These studies have emphasized the role of capsule during invasive infections. Capsules are generally considered to be antiphagocytic, since they inhibit the deposition of opsonizing complement and antibody onto the bacterial surface [5]. For Spn, different CPS types vary in their ability to block opsonophagocytic clearance, which is facilitated once CPS type-specific antibody is generated [6]. Invasive infection, however, is a biological 'dead end' for Spn, whose lifecycle depends on colonization of the upper respiratory tract (URT) of humans (the carrier state) and transmission from one carrier to another.
The biosynthesis of a thick capsule coat would be expected to confer a substantial metabolic burden in proportion to the amount of CPS expressed, which must be offset by a biological advantage. In vivo studies suggest that capsules enhance Spn retention along the URT epithelium by limiting bacterial binding to mucus and, thereby, preventing mechanical clearance via mucociliary flow [7]. However, many isolates of Spn and other members of the diverse Streptococcus mitis group are nonencapsulated [8], yet successfully colonize the upper respiratory tract (URT) as commensals [9][10][11]. Capsule also inhibits Spn adherence to epithelial cells cultured in vitro, raising the question of how encapsulated organisms persist on mucosal surfaces [12][13][14][15].
It has been suggested that many encapsulated species deal with these potential advantages and disadvantages of capsule by variation between forms with high and low levels of CPS expression. For many Spn, this variation may be observed as on-off switching or phase variation between opaque and transparent colony forms, which express thick and thin capsules, respectively, and differ both in their abilities to colonize and cause invasive infection [16]. A molecular switch involving spontaneous recombination events affecting genome-wide methylation is now known to control Spn phase variation [17][18][19]. However, because this mechanism affects many bacterial characteristics besides CPS amount, phase variation in opacity is not a specific means to determine the consequences of CPS amount diversity. There is also evidence that capsule expression is dynamic within the respiratory tract [13] and that it may shed in response to the epithelium [20].
The purpose of this study is to use defined mutants differing only in the amount of CPS to reassess the specific contributions of capsule in Spn-host interactions. These are tested in an infant mouse model that recapitulates key aspects of early childhood infection, including prolonged carriage, direct transit from the mucosal surface of the URT to bloodstream (occult bacteremia), bacteremic infection, and host-to-host transmission.

Characterization of strains expressing different amounts of CPS
A type 6A clinical isolate (P376) was screened to identify spontaneous mutants expressing different amounts of CPS based on altered colony morphology (Table 1). Two candidates, P384 and P385, previously shown to express~45% and~40%, respectively, of WT levels of type 6A CPS using a quantitative capture ELISA were further analyzed by whole genome sequencing [21]. In comparison to the parental strain, both strains contained a missense mutation in the cps sugar transferase gene cpsE. In addition, both strains contained a second point missense mutation in an unrelated locus. To confirm that only the former mutation was responsible for altered CPS expression, the WT cps region was used to correct the cpsE point mutation in T6A 40% , resulting in a corrected mutant (T6A CM ), identified among transformants by screening for WT colony morphology and validated by sequencing. After confirming restoration of full CPS expression by immunoblotting (Fig 1A), a functional assay was used to compare the relative ability of the strains to shield the bacterial surface from antibody binding to a conserved epitope underlying the capsule. As expected, strains with higher CPS expression were more protected from the binding of mAb TEPC-15 to phosphorylcholine, a feature of Spn teichoic acids, as measured by flow cytometry (Fig 1C). Differences in surface shielding between T6A 100% and T6A 40% were similar when grown in vitro or obtained from URT lavages of colonized pups and analyzed ex vivo.
Our analysis also included a previously described set of strains expressing different amounts of the type 4 CPS [22]. An unencapsulated mutant (P2422) containing the Sweet Janus cassette inserted in cps was used as recipient to restore the WT locus and to generate constructs with modified cps protomer regions expressing~20% or~50% of WT levels of type 4 CPS. We confirmed differences in type 4 CPS expression by immunoblotting ( Fig 1B) and then compared

PLOS PATHOGENS
Capsule amount in pneumococcal pathogenesis

PLOS PATHOGENS
Capsule amount in pneumococcal pathogenesis their ability to shield the bacterial surface from binding of mAb TEPC-15. Strains expressing 0% or 20% of CPS exhibited no shielding, whereas strains expressing 50% or 100% were equivalently shielded (Fig 1D). This suggests a threshold for effective shielding between 20% and 50% of WT levels of T4 CPS.

Role of CPS amount in invasive infection
The virulence of the strains was then compared following intraperitoneal (IP) challenge of a low dose (100 CFU) in 10-day old mice. For the type 6A strains, the humane endpoint was reached by 24 h post-inoculation (p.i.) for all pups given T6A 100% or T6A CM and blood cultures showed high levels of bacteremia (Fig 2A). In contrast, none of the pups challenged with the mutants expressing T6A 40% appeared septic or had detectable bacteremia. For the type 4 constructs, bacteremia was only detected in pups challenged with the fully encapsulated strain ( Fig  2B). These results demonstrate the contribution of capsule to virulence in this model and show a relationship between amounts of CPS and the capacity to sustain bloodstream infection. Because mice are naïve hosts and lack naturally-acquired anti-Spn antibodies, complement is the main serum opsonin. Complement deposition by the strains was compared using flow cytometry by detection of C3b bound to the bacterial surface following incubation with normal mouse serum. Similar to the binding of mAb TEPC-15, lower CPS expression (e.g. T6A 40% and T6A 45% ) was associated with increased C3b deposition ( Fig 1E). (Subsequently, further analysis of T6A 45% was discontinued due to the established in vivo role of ciaR and the potential confounding effects of the point mutation in this gene [23]) The pattern of C3b deposition for the type 4 constructs paralleled mAb TEPC-15 binding with a threshold between 20% and 50% of WT levels of CPS ( Fig 1F). Thus, the amount of CPS correlated with inhibition of both cell surface antibody binding and complement deposition.
To confirm that complement deposition was responsible for the attenuated virulence of mutants with lower amounts of CPS, the IP challenge was repeated in pups in which serum complement was depleted by prior treatment with cobra venom factor. In pups lacking complement activity, strains T6A 40% and T4 50% were able to cause high level bacteremia unlike in complement-sufficient mice (Fig 2C and 2D). Together these findings demonstrate that only by the expression of high levels of CPS is Spn able to shield its surface from opsonins that would otherwise promote its clearance during bloodstream infection.

Role of CPS amount in colonization dynamics
We then considered how amounts of CPS affect colonization. Mice at 10 (type 6A) or 4 (type 4) days of life were given an intranasal (IN) inoculum (10 2 CFU), and the numbers of colonizing Spn were assessed in quantitative cultures of nasal lavages over a 28-day period (Fig 3A). At the initial time point (day 1 p.i.), the density of colonizing Spn was significantly lower for the T6A 40% compared to both the parent and corrected mutant. This difference was maintained throughout the 28-day time course, by the end of which most mice cleared the less encapsulated mutant. For the type 4 constructs, colonization density generally correlated with amounts of CPS over the time course and was significantly reduced for the strains expressing 0% and 20% of WT levels of CPS ( Fig 3C). Only these two constructs were completely cleared by the final time point.
The dynamics of colonization density, therefore, showed two distinct effects of CPS amount for both type 6A and 4 strains. In the early phase (day 1), differences in colonization density correlated with CPS amount, and this effect persisted through 14 days p.i. In later phase, there was a more precipitous decline in colonization density from 14-day p.i. for the T6A and T4 strains expressing less CPS that ultimately led to clearance.

PLOS PATHOGENS
Capsule amount in pneumococcal pathogenesis

Fig 2. Effect of differences in CPS amount on invasive infection. (A-B)
10-day-old pups were challenged intraperitoneally with Spn (~10 2 CFU) of (A) T6A or (B) T4 strains and sacrificed 24 hrs later when blood was collected for quantitative culture to determine bacterial burden in the bloodstream. Statistical significance was determined using a Kruskal-Wallis test followed by Dunn's correction. (C-D) Pups were pretreated with either cobra venom factor (CVF) or vehicle control (PBS). Limit of detection (LOD). Statistical significance was determined using the Mann-Whitney test. https://doi.org/10.1371/journal.ppat.1011509.g002

Capsule amount in pneumococcal pathogenesis
To further examine the early effect, we measured colonization density at 4 hrs p.i. Prior studies established that this timepoint is before bacterial replication becomes the main factor driving bacterial numbers, and, therefore, CFU counts largely reflect the retention of the inoculum in the URT [24]. Retention correlated with the amount of CPS and was significantly impaired for the T6A 40% and T4 0% mutants compared to more fully encapsulated strains of the same type (Fig 4A and 4B). To control for potential host-related differences, the 4-hour  . Statistical significance was determined using Kruskal-Wallis test followed by Dunn's correction. (C) Competition assay of T6A strains. Pups at 5-10 days of age were challenged IN (~10 2−3 CFU/strain) with an equal mixture of the two strains indicated below and sacrificed 4 hours later. URT lavages were collected for quantitative culture with strains distinguished by colony morphology. Values represent the ratio of strains in the lavage (output) over the ratio in the inoculum (input) for each pup. Statistical significance was determined using the Mann-Whitney test in comparison to a hypothetical value of 1 (representing no competition). https://doi.org/10.1371/journal.ppat.1011509.g004

PLOS PATHOGENS
Capsule amount in pneumococcal pathogenesis retention assay was repeated in a competitive assay in which equivalent numbers of T6A 100% or T6A CM competed with T6A 40% were delivered to the same pup ( Fig 4C). Both fully encapsulated strains outcompeted the point mutant expressing less CPS. These results demonstrate that the amount of CPS affects initial interaction of Spn and the host.
To further examine the later effect, we considered host pathways known to mediate clearance of colonization. Prior studies showed this requires IL-17-dependent recruitment of professional phagocytes, as mice lacking the common receptor for IL-17 cytokines exhibit impaired phagocyte recruitment and delayed clearance [25,26]. The more accelerated clearance in WT mice of T6A 40% was absent in IL-17ra -/mice ( Fig 3B). This observation shows that amounts of CPS affect IL-17-dependent clearance mechanisms.

Role of CPS amount in other host interactions
The type 6A isolate used in this study is capable of causing bacteremia and sepsis following URT colonization (occult bacteremia) [27]. Thus, by comparing survival following IN challenge, we could assess the overall effects of amounts of CPS, since this outcome involves the steps of 1) retention of Spn in the URT, 2) establishment of colonization, 3) transit from the mucosal surface to bloodstream, and 4) proliferation in the bloodstream. When tested in 10-day old mice, the humane endpoint was reached beginning 2 days p.i. (Fig 5). The point mutant expressing less CPS (T6A 40% ) was avirulent and contrasted with the parent and corrected mutant, both of which caused sepsis in about half the mice.
We then analyzed the effect of CPS amount on the rate of intra-litter pup-to-pup transmission. This was carried out in the setting of influenza A co-infection administered to all pups in order to increase the frequency of transmission, thus allowing for meaningful comparisons [28]. Acquisition of infection by contact pups from Spn colonized index pups was proportional to the amount of CPS (Table 2). Transmission rates were significantly reduced for type 6A (40%) and type 4 (0 and 20%) mutants compared to strains expressing higher levels of CPS.

PLOS PATHOGENS
Capsule amount in pneumococcal pathogenesis

Comparison of CPS amount in clinical isolates
Lastly, we used immunoblotting to compare CPS amount among clinical isolates of the same type, but differing in date, site and location of collection. Nine type 6A isolates, including P376 analyzed above, varied by as much as 65-fold in the amount of CPS under standard growth conditions (Fig 6). For illustration purposes, both opaque (O) and transparent (T) forms of P461 were included and showed the expected difference of~10-fold. These differences in CPS expression were not limited to type 6A. Isolates of two other common Spn types 6B (n = 11) and 23F (n = 10) also showed 35-and 6.6-fold variation, respectively, in amounts of CPS within a given type. This limited survey demonstrates that CPS amount is a highly variable feature of Spn.
To further explore effects of capsule amount during infection, one of the type 6A clinical isolates expressing a relatively low amount of capsule was selected. P592 was unable to cause bacteremia following IP inoculation at a low dose (10 2 CFU) as expected (Fig 7A). Following  were grown to mid-log phase and a lysate adjusted for equal loading was applied to a nitrocellulose membrane. CPS was detected with type-specific antisera and relative binding compared in relation to a standard curve generated using purified CPS of the same type (shown boxed with amount loaded indicated) and quantified by densitometry. The level of CPS of each isolate is expressed relative to the strain of the same type with the least CPS (1x). An unencapsulated mutant served as negative control (Δcps). https://doi.org/10.1371/journal.ppat.1011509.g006

PLOS PATHOGENS
Capsule amount in pneumococcal pathogenesis IP challenge with a high dose (10 7 CFU), a variant P2797, with a colony phenotype of increased opacity, was obtained from blood culture of a single pup (Table 1). In contrast to its parent, P2797 was able to cause bacteremia following challenge at a low dose (10 2 CFU). Immunoblotting showed that P2797 expressed 11-fold more CPS relative to its parent (Fig 7B). Whole genome sequencing and comparison to P592 revealed that these changes were associated with acquisition of a Thr321Ala missense mutation in CpsE of P2797. P592 colonized pups at relatively low levels ( Fig 7C). The selection for this mutation that increased CPS expression in P2797 allowed for more robust colonization. Together, these observations add to the evidence that capsule amounts permissive for invasive infection enhance colonization.

Discussion
This study examined the effects of differences in amounts of the major virulence determinant of a leading pathogen. Defined, isogenic mutants carrying a single spontaneous mutation in the cps locus (T6A) or strains with changes to the cps promoter region (T4) were compared. Amounts of CPS per bacterial cell based on immunological assays with type-specific antibodies were used as a proxy for capsule thickness. Results confirm that thick capsules are required to inhibit i) binding of antibodies to underlying conserved surface antigens and ii) deposition of serum complement. These characteristics correlated with the capacity to evade opsonization, sustain bacteremic infection, and cause sepsis following invasion from the URT. However, the characteristics promoting invasive infection would not explain why Spn expresses thick capsule, as it is a 'dead end' for the organism. Thus, we investigated how CPS amount factors into the critical aspects of its commensal lifestyle; establishment in the URT (retention), stable colonization (density and duration), and transmission from host-to-host. CPS amount alone was shown to be important for each step in the dynamics of Spn colonization (retention, density and duration), which in turn impacts the chances of successful transmission. Although our

PLOS PATHOGENS
Capsule amount in pneumococcal pathogenesis study focused exclusively on infant models, our findings could be relevant to infection at later ages, including in the elderly, who also have a high burden of Spn infection. Several aspects of the mechanism involved in these observations merit further comment. WT T6A and T4 strains expressed amounts of CPS that effectively shielded the bacterial surface from potential opsonins and were permissive for bacteremic infection. Threshold amounts of CPS associated with these characteristics, however, appeared to differ between the two serotypes tested (>45% for T6A and <50% for T4). Thus, the relative importance of amounts of CPS must be considered separately for any given Spn type, since the physical properties and chemical composition of CPSs vary. In this regard, Magee and Yother previously examined the requirement for capsule during murine colonization and invasive infection [29]. They concluded that isolates producing reduced levels of capsule remain capable of colonization and causing invasive infection. Their study, however, depended on type 3 strains, which are unusual among pneumococci because they produce highly mucoid colonies. While type 3 strains, which express very thick capsules compared to other types, retain fitness in vivo with 20% of WT levels of CPS, the findings here suggest this is generally not the case. Additionally, we previously showed that unencapsulated Spn demonstrate an impaired ability to escape mucus binding within the loose mucus layer during the first minutes after arrival in the URT [7]. This finding indicated that capsule could inhibit initial clearance which occurs by the mechanical removal of particles embedded in loose mucus by mucociliary flow. Entrapment in mucus, which is also facilitated by the agglutinating effect of specific mucosal antibody, prevents establishment along the epithelial surface where stable colonization occurs [30]. Results in this report show that similar to unencapsulated mutants, strains expressing reduced amounts of CPS are also more susceptible to rapid removal, as seen with decreased initial retention in the URT [7]. This effect correlates with previous findings using the T4 strains analyzed here showing that mucus binding in a plate assay inversely correlates with amounts CPS-a result that could be explained by repulsive hydrostatic and charge properties of CPS [22]. A thicker capsule containing more CPS, therefore, appears to enhance mucus evasion and could explain why it is advantageous despite the metabolic cost for its biosynthesis. This also suggests that CPS-mediated evasion of mucus offsets any negative effects of capsule in adhering to host cells as seen in tissue culture models [13].
Early success in establishment on the URT mucosal surface has several consequences for the organism. We have shown that the Spn that initially colonize their niche have a substantial competitive advantage over those arriving later due to the early production of quorum sensing-dependent anti-Spn fratricidal effectors [24]. Additionally, when comparing isogenic constructs of different serotypes (capsule-switch mutants), we observed that a competitive success during the first hours of establishment in the host correlated with colonization duration over many weeks [26].
Similarly, the effect of increased CPS amount in facilitating initial success in the URT correlated with long term persistence. The effect of CPS amount on sustaining high colonization density and in prolonging colonization duration was shown here to be dependent on IL-17 signaling. This could be explained by IL-17-dependent recruitment of professional phagocytes [25]. Thicker capsules could inhibit the non-opsonic uptake by immune cells by macrophage scavenger receptors such as MARCO during colonization [31]. It should also be noted that in mouse studies neither complement nor specific antibodies generated during colonization appear to contribute to URT clearance once colonization is established [32,33]. This provides further evidence that the capsule-mediated shielding against the deposition of opsonins may not be the capsule's primary function during commensal host interactions. This is in contrast to invasive infection, where the effect of capsule amount in blocking the deposition of complement correlated with Spn's ability to survive in the bloodstream. For those pneumococcal types

PLOS PATHOGENS
Capsule amount in pneumococcal pathogenesis cleared from the bloodstream in a complement-independent manner, it would be interesting to examine the effect of capsule amount on their direct interaction with phagocytes, including liver-resident macrophage Kupffer cells [34].
Increased CPS amount also correlated with the rate of transmission from pup-to-pup. We previously showed that more CPS per strain enhances T4 Spn shedding, a rate limiting step for transmission in this model [22]. Shedding was not considered separately in this report, since colonization density was affected by capsule amount and is a main determinant of the number of shed Spn. Transmission also requires robust colonization. Since CPS amount affected colonization density over the time period during which contact and index pups were together, we cannot distinguish an independent effect on transmission apart from that on colonization. Also, because of differences in colonization dynamics, we could not distinguish whether differences in transmission were due to altered shedding from the index pups or acquisition and retention by contact pups.
The isogenic mutants tested here varied up to 5-fold (20% to 100%) of WT amounts of CPS. When clinical isolates of three serotypes were compared, we observed a far greater range of CPS amounts (up to 65-fold). Some of these differences could be due to opacity phase variation, which previously was reported to account for up to a 5.6-fold difference in capsule amount [21]. Many of the type 6A clinical isolates expressed less than 40% of the level CPS of P376 (T6A 100% ), which in the animal model was well below the threshold amount required for invasive infection, robust colonization, and efficient transmission. This wide range of variation was not unexpected considering variations in colony morphology, ranging from rough to mucoid, among different isolates. It is now clear that multiple factors contribute to the variation in CPS expression among isolates, including opacity phase variation, differences in cps promoter strength, a cis-acting regulatory element, multiple transcriptional factors, translational control by environmental conditions such as temperature, and post-translational control by tyrosine phosphorylation of CpsD [21,[35][36][37][38][39]. As the current report shows that much smaller differences in CPS amounts (<5-fold) affect each aspect of Spn host interactions, variation in this bacterial factor should be taken into account when comparing the pathogenicity of different isolates.
Finally, since many of the bacterial species found on mucosal surfaces express encapsulated forms, our findings demonstrating how CPS amount affects colonization and transmission dynamics may be relevant to other species.

Ethics statement
All animal experiments followed the guidelines summarized by the National Science Foundation Animal Welfare Act (AWA) and the Public Health Service Policy on the Humane Care and Use of Laboratory Animals. The Institutional Animal Care and Use Committee (IACUC) at New York University Grossman School of Medicine oversees the welfare, well-being, proper care and use of all animals, and they have approved the protocol used in this study (IA16-00538).
C57BL/6J WT mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and congenic IL17ra -/knock-out mice were acquired from Amgen Inc. Mice were bred and housed in a conventional animal facility. Pups were maintained with their dam until weaning at age 3 wk. Weaned mice were fed ad lib the PicoLab Rodent Diet 20, a 20% protein diet formulation, and were given acidified water for consumption. Additionally, the animals were kept on a light-cycle of 12 hours on, 12 hours off with a temperature in the animal facility of 70˚F (±2˚F). Pups were monitored daily for signs of sepsis.

Bacterial culture and strain construction
Types 4 and 6A strains used in the study are described in Table 1. Clinical isolates were confirmed as Spn by sensitivity to ethylhydrocupreine hydrochloride (optochin) and typed using specific antisera (Statens Serum Institute). Pneumococci were grown statically in tryptic soy (TS) broth (Becton, Dickinson) at 37˚C. Upon reaching the desired OD 620nm of~1.0, cells were washed and diluted in sterile phosphate-buffered saline (PBS) for inoculation. For quantitative culture, serial dilutions were plated on TS broth agar supplemented with an appropriate antibiotic and either 5% sheep blood or catalase (6,300 U/plate; Worthington Biochemical Corporation) and incubated overnight at 37˚C with 5% CO 2 .
Colonies expressing different amounts of CPS were selected by visual screening using microscopy with oblique, transmitted illumination as described [16]. Mutants were confirmed by whole-genome sequencing. A corrected mutant (P2752) of the cpsE mutation in P385 was constructed by transforming P385 as previously described [26] with the cps PCR product of P376 using primers 5'-ACC ATT GTC TCT ACC TCT CAC -3' and 5'-CGG AAT TCC TGT AAT TGA TGT CAT -3'. The transformation was verified using sequencing primer 5'-GAA GAT TCT CCT ACT TAC AGC AAC -3.

Whole-genome sequencing and genomic analysis
DNA libraries for strains P376, P384, and P385 were sequenced on an Illumina NovaSeq 6000 instrument to a coverage of at least 560x, producing 150 bp paired reads. The software fastp v0.20.1 was used with default settings to trim low-quality bases, remove adapters, and filter out low-quality reads [40]. The resulting trimmed, filtered reads of each isolate were aligned to an annotated genome assembly of Spn strain 6A-10 (RefSeq accession number: GCF_013047165.1) using Snippy v4.6.0 (https://github.com/tseemann/snippy/). The Snippy command snippy-core was used to compute a core-genome alignment and to call single-nucleotide variants (SNVs). The resulting core genome alignment had 2,007,997 positions, of which 3,072 were variable.
Accession number(s): The sequencing reads generated for this study are available in NCBI under BioProject PRJNA930766.

Immunoblotting
To visualize the differences in CPS expression, strains were grown to OD 620nm of 1.0 and an aliquot saved for total protein determination. 1 mL of culture was collected and then resuspended in PBS before lysis with 0.2% Triton-X 100 at RT. To 180 μL of lysed cells, 2 μL of proteinase K (50ug/ul) and 28 μL of 1X PBS were added to degrade cellular proteins at 65˚C for 15 min. The samples were then diluted 1:100 in 1X PBS and sonicated for homogenization at 60 Hz for 6x10 seconds. To perform the blot, a vacuum-slot blot device was used with sample loading adjusted based on bacterial density or total protein as indicated. Samples were loaded onto a nitrocellulose membrane alongside a purified CPS standard (Merck) of the same Spn serotype. The membrane was blocked in 5% milk in PBST for 30 min and then incubated with type-specific rabbit antiserum (Statens Serum Institute, 1:8000 to 40000) for 1 hr at RT. An unencapsulated strain, P2422, was incubated with type-specific antiserum to absorb out non-CPS specific binding. After washing the membrane, it was incubated with HRP-conjugated IgG goat anti-rabbit antibody (1:5000) for 1 hr at RT. The membrane was then developed with Thermo Femto Super sensitivity substrate and imaged using the iBright imaging system. Chemifluorescence readings were processed and densitometry area-under-the-curve analysis were performed in FIJI.

Bacteremia model
10-day-old pups of both sexes were given an IP inoculation containing 10 2 CFU of Spn suspended in 40 μl of PBS, as described previously [27]. Pups were euthanized 24 hrs post-inoculation, and blood was collected via cardiac puncture and cultured to assess for bacteremia. To select for a variant expressing increased CPS, the inoculum was increased to 10 7 CFU. To verify its importance in invasive infection, complement was depleted in 9-day-old pups of both sexes by IP administration of 20 ug of cobra venom factor (or PBS as a vehicle control) (Sigma-Aldrich) in 20 μL in PBS. The pups were then inoculated with Spn in the same manner as described above 24 hrs later. At 24 hrs post-inoculation, the pups were euthanized to perform blood collection and culture.

Colonization model
Pups of both sexes were given an intranasal inoculation without anesthesia containing 10 2 CFU of Spn suspended in 3 μl of PBS, as described previously [41] at either 4 days of age (T4 strains) or 10 days of age (T6A strains). To measure colonization density, pups were euthanized at the indicated time point by CO 2 asphyxiation followed by cardiac puncture. The URT was lavaged with 250-500 μl of sterile PBS from a needle inserted into the trachea, and fluid was collected from the nares for quantitative culture. For strains not resistant to streptomycin (200 ug/ml), neomycin (5 ug/ml) was used to select against contaminants.
To assess Spn retention, pups were euthanized at 4 hrs post-infection following an inoculum of 10 2−3 CFU. To evaluate in vivo competition at 4 hrs, pups were co-infected with two strains expressing different amounts of CPS (~10 2−3 CFU per strain). Pups were euthanized at 4 hrs post-infection, and URT lavages were collected and plated on TS agar plates with catalase and appropriate antibiotics. Numbers of each strain were obtained based on morphological differences in colonies. Competition indices were calculated as the ratio of strains in the output divided by the ratio of strains in the inoculum.
To compare invasive infection following intranasal challenge, pups were inoculated intranasally at the dose and age described above. Pups were monitored daily for survival or moribund endpoint for 14 days following inoculation.

Comparison of surface shielding
Bacterial strains grown in TS broth at 37˚C to an OD 620nm of 1.0 were washed and diluted in 1% PBS-BSA to a final concentration of 10 6 CFU in 100 μL. Bacteria were stained with the mAb to phosphorylcholine, TEPC-15 (Sigma-Aldrich, 1:5000 dilution) followed by a FITClabeled antibody to murine IgA. Reactions were carried out at 4˚C for 30 mins without spin or wash steps in between. After staining, samples were fixed with 4% paraformaldehyde (PFA), washed once with PBS, and resuspended in PBS. The surface shielding assay was also performed using ex vivo samples of bacteria collected from colonized mice. T6A strains P376 or P385 were administered IN to 10-day-old pups at a dose of 10 6 CFU in 3 μL. Two hours postinoculation, pups were euthanized, and URT lavages collected. Lavages were pooled from at least 6 pups, pelleted and resuspended in 100 μL and treated as described above. Samples were analyzed by flow cytometry using the LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star).

Comparison of complement binding
Bacterial strains grown in TS broth at 37˚C to an OD of 1.0 at 620nm were washed and resuspended in 1X PBS. 2.5x10 6 CFU of Spn in 25 μL were incubated with an equal volume of fresh

PLOS PATHOGENS
Capsule amount in pneumococcal pathogenesis normal mouse serum at 37˚C for 30 min. For negative controls, serum was pre-treated at 57˚C for 30 min to heat-inactivate complement. The incubation was ended by adding 50 μL of 20 mM EDTA in 1X PBS. Cells were pelleted, resuspended, and then stained with FITC-conjugated anti-mouse C3 antibody (MP Biomedicals) at 4˚C for 30 min. Cells were then fixed in 4% PFA and resuspended in 20 mM EDTA in 1x PBS. Samples were analyzed by flow cytometry using the LSRII flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star).

Transmission model
The pneumococcal transmission model with influenza A (IAV) co-infection was described in previous studies [42]. Briefly, one in four to five pups in the litter was randomly selected and, at an age of 4 days, infected with Spn (index mice) and then returned to cage containing the dam and the other uninfected pups (contact mice). For experiments involving type 6A strains, all pups in the litter were inoculated intranasally with 3 μl IAV/HKx31 containing 250 plaqueforming units three days after Spn infection. For experiments with type 4 strains, all pups were inoculated with IAV four days after Spn infection. Then, all pups were euthanized and nasal lavages were collected either four days after IAV infection (T6A strains) or six days after IAV infection (T4 strains). Lavages were cultured to detect bacterial transmission from the index to contact pups.